Chemical constituents, antioxidant, and antimicrobial activity of Allium chinense G. Don

Allium chinense G. Don is a medicinal and aromatic herb belonging to the family Amaryllidaceae, characterized by a high saponin content. The previous report has mostly been focused on the bulb, and there is very limited work on the leaf. The information about biological and chemical constituent of A. chinense leaf is still inadequate in contrast to the investigations reported on the bulb. To the best of our knowledge, there is no report on the hexane extract of both bulb and leaf extract. Therefore, the present investigation was focused on identifying and characterization of the hexane extracts of A. chinense bulb and leaf quantitatively and by using the GC-MS method and to know its scavenging, antibacterial, and antifungal activity. Twenty-eight bioactive compounds were identified in the bulb and nine in the leaf extract by GC-MS analysis. The versatile compounds present in the bulb are 2-methyloctacosane (21.30%), tetracontane (14.05%), eicosane, 10-methyl (12.06%), heneicosane (8.46%), octadecyl trifluoroacetate (6.48%), and 1-heneicosanol (5.76%), whereas in the leaf, it was phytol (35.76%), tetratetracontane (18.49%), perhydrofarnesyl acetone (14.76%), and heptadecane, 2,6-dimethyl (10.79%). In quantitative estimation, saponins were estimated to have the highest with 375.000 ± 0.577 mg/g in the leaf and 163.750 ± 0.433 mg/g in the bulb. The DPPH antioxidant scavenging activity was found to be minimum in both the bulb (IC50 = 678.347 μg/ml) and leaf (IC50 = 533.337 μg/ml). A. chinense extracts of both leaf and bulb exerted potential antibacterial effects against Staphylococcus aureus and Pseudomonas aeruginosa. Leaf extract exhibited greater antifungal activity than the bulb against Aspergillus niger. From the analysis, the hexane leaf extract exhibited higher antibacterial, antifungal, and antioxidant activity than the bulb. Their superior activity might be due to the higher content of total saponin and terpenes. This result will lead to further in-depth research towards the potential use of this plant; the bio-constituents can be further isolated and used in medical and therapeutic applications.


Background
Plants and their bioactive compounds are a potential source of medicine and are suitable with the prevailing demands for safe and effective treatment. Traditional knowledge procured over years of observation and interaction with the environment is a substantial source of modern medicine. Isolated and purified bioactive constituents of the plant can be used for drug development in pharmacology and medicine.
The genus Allium belongs to the family Amaryllidaceae has 500 species and are characterized by their rich content of organo-sulfur as their main bioactive compounds [1]. Allium chinense (Fig. 1) is cultivated in the North-Eastern part of India region; the whole plant is edible raw or cooked and is used in culinary as a flavoring agent. The plant has a strong onion-like aroma and pungent taste, the entire plant is an expectorant, carminative, and astringent also used to treat bronchitis, diarrhea, and angina pleurisy [2]. The bulb has high medicinal value in traditional Chinese medicine for over thousands of years and is the major source of the "Xiebai" drug used in treating thoracic pain, stenocardia heart, asthma, and stagnant blood [3]. Among the many bioactive compounds present in A. chinense, the important active compounds are steroidal saponins, amino acids, sulfur, nitrogen, and flavonoids compound [4]. The earlier study on A. chinense leaf essential oil reported 34 organo-sulfur-containing compounds accounting 94% of the total volatiles [5]. The chemical investigation carried out in essential oil of the bulb reported a profuse content of sulfide compounds, as high as 97.71% [6]. Spirostanol saponins extracted from 60% aqueous methanol extract [7] and laxogenin extracted from hot water extract [8] from A. chinense bulb have anti-tumor property. The protective effects of steroids from 60% ethanol bulb extract of A. chinense have great potential to prevent cardiac injuries induced by oxidative stress [9]. On literature survey, most of the earlier works have been carried out on the bulb and the biological and chemical report on A. chinense leaf is still scarce. However, no report to date has been found on hexane leaf and bulb extract of A. chinense. Therefore, the objective of the study was to characterize the chemical profile of the bulb and leaf hexane extract using GC-MS and the second objective was to determine the scavenging activity and antimicrobial activity of A. chinense.

Collection of plant material
Fresh and healthy plants of A. chinense were collected from the farmers in Kohima district, Nagaland, India. The collected plants were washed, carefully shade-dried till all the moisture contents were removed and ground to a fine powder using an electric grinder and stored in an airtight glass bottle at room temperature 27 ± 2°C.

Extraction
A. chinense leaf and bulb were extracted separately using a soxhlet extractor with 200 ml hexane for 18-20 h. The extracts were separated from the solvent using a rotary evaporator at 30-40°C and stored at 4°C for further use.

Quantitative analysis Estimation of total alkaloid content
The total alkaloids were determined using the method described by Tan [10]. Atropine was used as a standard for plotting the calibration curve. The extracts (1 mg/ml) were dissolved in 2 N HCl and washed with chloroform, the layers were separated using a separating funnel, later bromocresol green solution and phosphate buffer (pH 4.7) were added and the mixture was vortexed, the yellow color complex formed at the bottom was pipetted out to measure the absorbance at 470 nm. The total alkaloid content was estimated using the linear regression equation obtained from the standard graph of atropine further mean ± SD (n = 3) was calculated and expressed as mg/g atropine equivalent (AE).

Estimation of total flavonoids
Estimation of total flavonoids content was done using the aluminum trichloride (AlCl 3 ) method with different concentrations of quercetin as standard [11]. Extracts of 1 mg/ml were diluted with distilled water and 5% NaNO 2 solution was added. After being incubated for 6 min, 0.15 ml of AlCl 3 10% solution was added followed by 2 ml of 10% NaOH, the final volume was adjusted to 5 ml using distilled water, and it was then incubated at room temperature for 15 min. Absorbance was read at 510 nm. All analysis was carried out in triplicate times and the total flavonoid content was calculated using linear regression equation obtained from the standard plot of quercetin and mean ± SD (n = 3) was calculated. The result was expressed as mg/g quercetin equivalent (QE).

Estimation of total phenolic content
The total phenolics were estimated with Folin-Ciocalteu's method [12]. Gallic acid at varying concentrations was used as standard; plant extracts (1 mg/ml), 5 ml of FC (1:10) diluted reagent, and 4 ml sodium bicarbonate (7.5%) were taken in a test tube and the mixture was shaken and incubated in the dark for 30 min at 20°C. The absorbance was read at 765 nm, all the analysis was performed thrice. The total phenolic content was estimated using the linear regression equation from the standard calibration curve further mean ± SD (n = 3) was calculated and expressed as mg/g gallic acid equivalent (GAE).

Estimation of saponins
The saponin content of the extracts was measured as described by Le et al. [13]. Quillaia was used to obtain the standard calibration curve. The sample (1 mg/ml) was mixed with 500 μL of 8% vanillin and 500 μL of 72% sulfuric acid and incubated at 60°C for 10 min after cooling to room temperature absorbance was recorded at 544 nm. The total saponin content was quantified using the linear regression equation obtained from the standard graph of quillaia further mean ± SD (n = 3) was quantified as mg/g quillaia equivalent (QE). All determination was carried out in triplicates.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay
The ability of A. chinense bulb and leaf extracts to scavenge free radicals was assessed according to the standard DPPH method [14]. Ascorbic acid was used as the standard and the assay was performed in triplicate. One milliliter of the varying concentrations (1-100 μg/ml) was added to 3.0 ml of freshly prepared DPPH (0.06 mM). The mixture was further incubated in the dark for 15 min at room temperature (27 ± 2°C). The decrease in the absorbance was measured at 517 nm using a UV-Vis spectrophotometer. The percentage of inhibition was calculated using the following formula: where A 0 is absorbance of control (DPPH) and A 1 absorbance of extracts The calibration curve for inhibition was prepared and IC 50 values were calculated.

Antimicrobial analysis by disk diffusion method
The antimicrobial activity of the bulb and leaf extract was screened by the agar disk diffusion method. Muller-Hinton agar (MHA) (Hi-Media) plates were swabbed with gram-positive Staphylococcus aureus (MTCC916) and gram-negative Pseudomonas aeruginosa (MTCC741) bacteria and fungi Aspergillus niger (MTCC281) in Sabouraud dextrose agar (SDA) plate [15]. Plant extracts of 1 mg/ml concentration were prepared in 0.1% dimethyl sulfoxide (DMSO). Four wells measuring 6 mm were bored in the inoculated media with a sterile corkborer. The disks were loaded with 30 μl of the extract and placed on the MHA and SDA plate. The Petri-plates were further incubated at 37°C for 24 h for bacteria and 22°C for 48 h for fungi. At the end of incubation, zones of inhibition (ZOI) were observed around the disk and measured with a transparent ruler in millimeters. Streptomycin (30 μg/ml) and fluconazole (1 mg/ml) were used as the positive control for bacteria and fungi respectively. 0.1% DMSO was used as a negative control.

GC-MS analysis
To obtain the complete chemical profile, analysis was performed using GC-MS (SHIMADZU QP2010S) system, with the Rxi-5Sil MS column; length 30 meters, and helium (99.99%) as a carrier gas at a flow of 1.00

Identification of compounds
The identification of chemicals was based on the peaks of the compounds at different mass-to-charge ratios. Results were obtained in accordance with the mass spectral library of National Institute of Standards and Technology, NIST-11. The unknown spectrum was compared with the standard spectrum existing in the database of the WILEY 8 library.

Statistical analysis
All the estimation was carried out in triplicates. The data obtained in this study were analyzed using a one-way analysis of variance (ANOVA) by using Prism V. 5.00 (Graphpad Inc. USA). The results were expressed as mean ± SE. Significance value (P) < 0.05 was selected as a point of minimum significance rate.

Quantitative analysis
Phytochemicals have been analyzed quantitatively. The total alkaloid, flavonoid, phenol, and saponin concentration of the bulb and leaf extract are presented in Table 1.

DPPH assay
The activity of A. chinense extract against free radicals is shown in Table 2 and Fig. 2. The A. chinense extract showed less scavenging capacity both in the leaf (IC 50 = 533.337 μg/ml) and bulb (IC 50 = 678.347 μg/ml) when compared to standard ascorbic acid (IC 50 = 55.118 μg/ml) with significant difference (p < 0.05).

Discussion
In the quantitative estimation of A. chinense bulb and leaf, high content of saponin was observed. Saponin possesses antibiotic, insecticidal, and fungicidal activity [16]. Steroidal saponins isolated from A. chinense bulb possess anti-tumor property [7]. Saponins are cytotoxic and inhibit the migration ability of B16 and 4T1 cells [4]. Laxogenin a saponin isolated from A. chinense bulb have anti-tumor property in stage-two lung carcinogenesis [8]. Therefore, A. chinense can be used as a potential plant for the extraction of saponin for medicinal and pharmaceutical use.
Alkaloids have been reported to have anticancer, antibacterial, antiviral, and antifungal activity [17]. Aclidinium bromide a drug from alkaloid is used to treat chronic obstructive pulmonary disease; atropine and scopolamine are alkaloid derivatives used in traditional medicine for treating asthma [18]. A. chinense showed a moderate content of alkaloids which  could be exploited for their pharmacological properties.
Low content of phenols [19] and flavonoids [20] indicate low antioxidant activity. Phenols are known to have anti-inflammatory, antimicrobial, anesthetic, antioxidant, anti-tubercular, anticancer, analgesic, and anti-Parkinson activity [21]. Flavonoids are known to have antioxidant effects and have been shown to inhibit the initiation and progression promotion of tumors [22]; consumption of flavonoids decreases coronary heart disease [23].
DPPH is a stable free radical with absorption spectra at 517 nm and loses its ideal absorption accepting an electron, resulting in a change from purple to yellow color, displaying the scavenging potential [24]. The low or negligible antioxidant capacity in the hexane leaf and bulb extract may be due to the low content of phenol [19] and flavonoid [20]. Our findings correlate with the previous report by Lin in A. chinense bulb [25].
The leaf extract showed higher antibacterial activity (20 mm) than the standard (18 mm) against P. aeruginosa, it is known that gram-negative bacteria are highly resistant to many antibiotics. The absence of ZOI was interpreted as the absence of activity. The activities are expressed as resistant if ZOI was less than 7 mm, intermediate (8-10 mm), and sensitive if more than 11 mm [26]. On observing the zone of inhibition, the bulb exhibited resistant activity whereas leaf was highly sensitive as an antibacterial. In A. niger, bulb extract showed intermediate (7 mm) activity whereas sensitive activity with 14 mm ZOI was observed in the leaf extract. The high composition of terpene, viz. phytol (35.76%) and perhydrofarnesyl (14.7%) in the leaf and 2-methyl octacosane (21.30%) in the bulb exhibited antimicrobial activity [19,24,27]. The leaf extract has an outstanding antibacterial activity against gram-negative bacteria P. aeruginosa.
The result of the current study supports the use of both bulb and leaf hexane extracts as a potential antibacterial and antifungal; the leaf extract presented as a wide antimicrobial spectrum compared to the bulb.
The GC-MS characterization revealed that the compounds found in both the bulb and leaves extract were not reported earlier in this plant. The difference in results reported in essential oil [6] may be due to the extraction method, soil pH, seasonal variation, climatic conditions, and many other factors.
In the leaf extract, phytol was the major component identified with a concentration of 35.76% and retention time of 20.475 min which is an acyclic diterpene with anticancer, anti-diuretic, nematicide, hepatoprotective, hypocholesterolemic, anticoronary, antiandrogenic antimicrobial, antioxidant, antiarthritic, anti-inflammatory, antidiabetic, and immunostimulatory [27] followed by tetratetracontane with a peak area of 18.49% appeared at 33.630 min is a long chain alkane, having antibacterial activity [28]. Perhydrofarnesyl acetone with 14.76% was the first compound detected in the leaf at 17.148 min and is a sesquiterpenoid that has extensive biological activities such as, antimicrobial, allelopathic, cytotoxic, and antifeedant activity [29].
The GC-MS analysis of the bulb displayed alkane as the major group. In the 28 compounds revealed, the dominance of compounds such as 2-methyloctacosane with the peak area of 21.30% appeared at 27.601 min which is an alkane with antimicrobial [30]. Tetratetracontane with a peak area of 14.05% which appeared at 30.497 min is an alkane having antibacterial property [28]. Eicosane, 10-methyl with a peak area of 12.06% appeared at 24.850 min, an alkane which has high antioxidant [31]. Heneicosane with a peak area of 8.46% reported at 33.939 min is an aliphatic hydrocarbon; it belongs to the higher alkane group having bio pesticidal property [32]. The GC-MS revealed that the bulb and leaves have a different bio-constituent; only three compounds tetratetracontane, nonadecane, and 2-methyl-octadecyl trifluoroacetate appeared in both the extract. Our findings on the chemical composition of hexane bulb and leaf extract did not correlate with the previous reports on the essential oil [5,6] and no sulfide-containing compounds were identified in the crude extract.

Conclusion
The quantitative analysis showed a high content of saponins in the leaf extract. GC-MS analysis of the extract suggests that numerous medicinally important bioactive constituents are present in A. chinense leaf and bulb, with high terpene content in the leaf and alkanes in the bulb.
These important findings revealed through our study suggest isolation of bioactive compounds, and screening its activity will have tremendous benefits, considering its availability, that it is edible and has medicinal value, and that it is non-toxic; however, a toxicological analysis would be of necessity to develop safe drugs. This work represents an initial step to understand the plant phytochemical constitution which could facilitate further investigation.